Cadaveric transplantation.R Gokal
Manchester Royal Infirmary, England, UK.
Transplantation is already the optimum treatment for terminal renal failure. Donor organ shortage means that there are large number of patients on dialysis awaiting this treatment. This has in some countries led to unacceptable unscrupulous practices of live non-related graft donation. The outcome of graft and patient after transplantation has improved significantly based on a better understanding of immunopathology, immunosuppression and tissue typing. The future is promising and xenografting may well solve the organ shortage but undoubtedly will raise other issues.
Keywords: Ethics, Medical, Forecasting, Histocompatibility Testing, Human, Immunosuppressive Agents, immunology,therapeutic use,Kidney Transplantation, immunology,methods,mortality,trends,Survival Rate, Transplantation Immunology, Treatment Outcome,
Renal transplantation is now accepted as the treatment of choice for patients with end stage renal failure. During the last decade both patient and graft survival rates have increased significantly and when assessed at one year are now greater than 90% and 80% respectively. These marked improvements have occurred at a time when increasing number of patients in the older age groups and with more complex forms of renal disease and casemix are being accepted for transplantation. The reasons for the improvement in clinical results are not really fully understood but are almost certainly linked with tissue typing policy, greater understanding of immunology and immunological monitoring, improvements in immunosuppression therapy and better clinical management of recipients. The main limitations in organ transplantation is the supply of cadaveric organs and in some parts of the world this has led to the exploitation of donor sources which would otherwise be judged inappropriate or ethically unacceptable; this is likely to continue in the forseeable future as demand will continue to outstrip supply.
Fra Angelico's famous picture of the Saints Cosmas and Damian grafting a black leg into a white patient is often shown as the first transplantation. Modern transplantation has not yet caught up with the reputed skill of Cosmas and Damian. Leaving the realms of speculation, transplantation could perhaps be considered to begin with a discovery in about 1860 by Reverdine and others that slivers of skin could be transplanted from one part of the body to another but failed if transferred from one individual to another. Carrel using improved methods of suturing carried out kidney grafts between cats and dogs and then removed the animals' own kidneys and felt that they functioned well for a few days after which urine ceased. He was given a Nobel Prize for his work. It was not until 1943 following Peter Medawar's demonstration of the basic immunological basis for organ transplant rejection (Nobel prize) early pioneers of human clinical organ transplantation attempted to ensure success either by transplanting between genetically identical individuals or by suppressing the vigorous host immune response. Murray in 1950 attempted transplantation in humans using cadaver kidneys (subsequently Nobel Prize) but rejection was still a major problem at it is today. To overcome this use of steroids, 6 mercaptopurine (by Elion in 1960 Nobel prize) and azathioprine (Roy Calne in the early sixties) was undertaken in setting the modern trend of post-transplant care. Further advances came about in the 1960s when a highly polymorphic human major histocompatibility system (HLA typing) was set out by Dausset (Nobel prize), which subsequently paved the way for further developments.
Tissue typing is the colloquial name for the blood group antigens of the nucleated cells. There are most easily demonstrated on the leukocytes and therefore they are known as the human leukocyte antigens (HLA). This system is controlled by a number of closely linked complex loci on chromosome 6 that define several different categories of biologically important antigenic markers. Among these are the Class I antigens, HLA-A, HLA-B and HLA-CW which are present on all nucleated cells of the body and can be regarded as the bodies antigenic "uniform" by which it identifies self from nonself, and the Class II antigens, HLA-DR, HLA-DP, and HLA-DQ which are present on B lymphocytes, Kupffer cells and activated T Cells and are involved in cell to cell communication. Each individual is diploid and therefore can have no more than two antigens coded by any one locus. Each individual has two chromosome 6 and in the production of germ cells, these separate. Each chromosome 6 carries a closely linked group of HLA markers, which is known as a haplotype: that is, the haploid expression of the HLA type. The antigens of one haploid type are conveyed as a unit en bloc to the offspring.
If a kidney is transplanted into an ABO incompatible recipient it is usually hyper-acutely rejected. For this reason kidney transplantation is usually only performed with an ABO compatible donors and recipients. Similarly if anti -HLA antibodies are present in the serum of a kidney recipient to an HLA-antigen of the donor, hyperacute rejection normally ensures. To try to avoid this, cytotoxic cross match of the serum of the graft recipient against the lymphocytes of the potential donor is carried out before transplant. If the crossmatch is positive that is if the recipient serum kills potential donor cells, the transplant is riot performed. This is true if the antibody is IgG of anti-HLA specificity. However, IgM autoantibody also causes cytoxicity under the normal test conditions but is believed to be of no clinical significance for graft surviva1. It is possible to discriminate between these two type of antibodies.
Serum samples of patients awaiting transplants are regularly screened by the cytotoxic tests against a panel of lymphocytes of normal individuals in order to define the strength, specificity and frequency of reactions of antibodies in the patients' sera. After the patient has been stimulated by HLA antigens, as occurs during pregnancy, blood transfusion and organ transplant, the anti-HLA antibody strength and frequency reaction against the panel may rise, but, in the, absence of stimulus, the antibody may decline with The "peak" serum sample, i.e. the one with the titre and greatest frequency of reaction may a positive cross match, while the current serum gives a negative cross match. What policy should be followed? The work of Falk et al  shows that if the current serum gives a negative cross match with the donor even though a historical serum in the past gives a positive cross match then survival for first grafts may not be detectably different from those with a historical negative and current negative cross match result. Many transplant centres ignore a historical positive cross match if the cross match with current serum is negative.
Centres advocating allocation of organs to recipients on 'the basis of HLA mismatch, site studies showing that transplant survival is significantly improved in cases where a high degree of HLA matching is achieved, over cases where there is little or no HLA match . In our own single centre study at the Manchester Royal Infirmary where allocation of cadaver kidneys has always been based on HLA matching, we find that those who had sero mismatches for HLA-DR specifities have a transplant survival of 85% at one year of follow-up which is 17.5% higher than complete HLA-DR mismatched recipient. The cases with one HLA-DR mismatch had intermediate survival [Table - 1]. The rationale for matching is not only to achieve good results but also to maximise a rare resource. The number of cadaveric organs available for transplantation has stabilised worldwide at approximately 20 per million population per year and this never meets the ever increasing demand. In such a situation it is essential to maximise the efficiency of the rare resource available and matching for HLA alleles and specifities cannot do anything but contribute positively to that. This does mean that individuals with rare phenotypes may have to wait longer for a suitably matched donor but limited effective matching perhaps just for HLA DR specificty is possible given that the HLA-DR polymorphism is less than HLA-A and HLA-B or all three loci combined.
The immune system is responsible for maintaining homeostatsis while protecting the body from the effects of foreign material or toxins. The branch of the immune system most commonly associated with the process of rejection is the lymphoid system, which is composed of lymphocytes that are primarily responsible for the recognition of antigen. There are three types of immune responses: humoral, cellular and combined humoral and cellular. The major function of humoral immunity, which is mediated by B lymphocytes is the production of antibodies. Cellular immunity, mediated by T-lymphocytes is associated with the interaction of an antigen and a sensitised lymphocyte. T- lymphocytes are subdivided into effector and regulator cells. Effector T-cells contain cytotoxic and memory substances, which attack antigens. Regulator T-cells contain helper and suppressive substances, which control the cytotoxic effects. Cytotoxic T-cells bind to target cells and facilitate their destruction through the release of lymphokines that stimulate an inflammatory response. IL-1 and IL-2 stimulate the proliferation of T-cells, which are important in the bodies defence against viral and fungal infections, mediate accelerated and acute rejection of transplanted organs.
Rejection of an allograft is a complex immune process that is only partially understood.  The first step of the immunological reaction involves the presentation of donor antigens to the patients' immune systems. Presentation to host T- and B- lymphocytes may be carried out by host antigen presenting cells (APC) after the donor antigen is processed or by donor APC directly. This stimulus activates the lymphocytes to divide and multiply, leading to a generation of both cell mediated and humoral effects. Precursor helper T-lymphocytes respond to the donor antigens and IL-1 derived from macrophages and other immunologic stimuli to produce other lymphokines such as IL-2 and interferon gamma. At the same time, precursor cytotoxic T-lymphocytes are stimulated by donor antigens to express receptors for IL-2. Once cytotoxic T-lymphocytes express IL-2 receptors, they are stimulated to multiply with the help of helper Tlymphocyte derived IL-2. These committed cytotoxic T-lymphocytes bind directly to cells of the transplanted organ and result in cell lysis. After the donor antigen is recognised, IL-2 produced by helper T-lymphocytes causes the release of T derived B-cell growth factors. This leads to multiplication and differentiation of B lymphocytes, intra-cellular immune globulin synthesis and antibody secretion. Antibodies primarily target the graft endothelium. Cell destruction is caused by activation of the complement cascade or by antibody dependent cell mediated cytoxicity.
Immunosuppressant drugs, a more thorough understanding of the mechanisms of rejection and the introduction of newer, more refined drugs have had a large impact on the survival of transplant grafts and patient. Immunosuppressive agents are listed in [Table - 2], which also shows their mechanism of action. In essence all anti-rejection agents block aspects of T-cell activation. The T-cell activation is a coordinated preprogrammed process. In non-pre-sensitised patients graft rejection is dependent on activation of allo-reactive T-cells and antigen presenting cells as described above. This is a T-cell dependent process that recruits a diverse assay of leukocytes.
The most interesting of the immunosuppsressive agents which has revolutionised organ transplantation is cyclosporin which was developed in the 1970s and has a much more specific immunological effect than corticosteroids or azathioprine. It acts by blocking calcium dependent T-cell activitation, thus reducing synthesis of IL-2. It may also have a direct inhibitory effect on IL-2 genetranscription via binding to cyclophylin.
The excellent success of cyclosporin as an immunosuppressant medication has not come without any adverse effects. Two of the more common adverse effects are hepatotoxicity and nephrotoxicity. The pathophysiology of these effects is not completely understood. The problem with drug induced nephrotoxicity is that it must be distinguished from ATN and rejection. This can be achieved by judicious use of isotope scanning, cyclopsorin blood levels and eventually renal biopsy.
This is the first therapeutic monoclonal antibody produced for use in humans. It was developed as means of suppressing T-cell- medicated rejectio.i. OKT3 is IgA2a immunoglobulin that binds to a surface antigen on T-lymphocytes. All mature T-cells express this antigen as well as a surface antigen. Once OKT3 is bound to a region of T-cell called CD3 they are thought to be coated and removed from the circulation by reticulo-endothelial system. Adverse effects of OKT3 include fever, tachycardia, chills, headaches, hypertension, nausea and vomiting. It is used mainly for acute rejection, which is resistant to steroids.
Antithymocytic globulin (ATG); and antilymphocytic globulin (ALG)
Before the advent of monoclonal antibody preparations polyclonal antibody preparations were available. Both these products can be used as induction immunosuppressive agents or as treatment for acute rejection or steroid resistant rejection.
New immunosuppsressive agents
Currently a number of other immunosuppressive drugs are in various stages of development. A new drug with the designation FK506 is undergoing therapeutic trials in renal transplantation. It is a naturally occurring macrolide antibiotic that was isolated from a Japanese fungus. FK506 has similar modes of action to cyclosporin. Other agents in various stages of development include rapamycin, anti-HULY-M2, 15 deoxyspergualin, RS61443.
Survival of kidney transplant is often expressed in actuarial curves, which show the graft surviving at times after transplant as a percentage of the total grafts carried out. The greatest loss of kidney grafts occurs during the first three months after transplantation. A large number of factors are relevant to survival of an allografted kidney. These include blood transfusion prior to grafting, presence of cytotoxic antibodies in the recipient, number of the allograft, the age, race of both donor and recipient, ischaemic time of the donor kidney, original disease of the recipient and method of preservation of the kidney as well as the more obvious factors such as skill of the surgeons, and clinical management of the immunosuppression of the patient post-transplant.
Over the last two decades overall transplant survival has greatly improved with advances in the immunosuppressive techniques and with experience gained by the transplant team.
Our own transplant survival data at the Manchester Royal Infirmary are shown in Fig.1, which shows that the graft survival over the 25 years of transplantation at the Centre has shown steady improvement with a current one year graft survival of around 95%. Over the same period of time the number of grafts that are performed with a zero DR-mismatch has increased to around 60%. The Unit follows a cyclopsorin monotherapy policy where all grafts that are immediately functioning will receive monotherapy whilst riple therapy of prednisolone, azathioprine and yclosporin are for those that are in tubular necrosis ,. Figures from the European Dialysis Transplant Registry also show dramatic: improvement in both patient and graft survival after the first cadaveric graft. Five years graft survival improved in the patients transplanted in 1986-87 (60%) as compared to those in 1980-82 (45%). Data are worse in diabetics at all ages by about 10% as compared to non-diabetics. Matching has significant impact on graft survival and the importance of a complete DR match (zero mismatch), which gives the best result, cannot be overemphasised. A mismatch at the B locus is not thought to be as important as the DR locus. Matching at the A locus is not significant for first grafts but for second and subsequent cadaveric grafts all loci have a bearing on graft survival. Mitsuishi and Terasaki  report on a 6 to 10% increase in one year graft survival for patients matched or mismatched for HLA A, B, DR, whilst the long term half-life of zero mismatched A-B-DR grafts was 10.7 years as compared to 6 antigen mismatch.
Ethics in the medical context relates to the obligations of a moral nature, which govern the practice of medicine, which relies on the duty to serve and protect the interest of the patient in ways consistent with the ethics of the profession and moral values held by society. Organ donation therefore especially in terms of the live donation should unmistakably be a charitable act arising from a special relationship between donor and recipient. All motives other than those associated with an unsolicited gift should be excluded. It is important to obtain informed consent from the donor in terms of related live donor, the offer should be voluntary with no coercion, the motive should be ultruistic and genetically related. In non related live donor, donor's doctor should ensure that the donor is a volunteer, no financial or other inducement has been given, and he understands the nature of the procedure, its consequences and risks, (eg death in 1/1600 of donor operations does occur). In this respect several countries have Transplant Acts like that in the UK, which prohibits any sale of donor organs.
In terms of cadaveric transplantation several countries have a code of practice, which includes transplantation check-list, criteria for brain death. In the UK all these are enshrined in the British Transplant Society recommendations and the Human Organ Transplant Act 1989 which makes it an offense to make or receive payment for the supply of an organ, advertisement relating to supplies and referring unrelated donors to an Unrelated Live Transplantation Regulatory Authority (ULTRA).
International agreement on organ donation is going to be difficult to arrive at because availability of organs and of dialysis services is important factors and ethical viewpoints vary throughout the world and is dependent on cultural, religious beliefs and socio-economic circumstances. Even though the World Health Organisation has set out a statement of guiding principles, it is important to realise that one cannot transport en bloc western attitudes and values to the rest of the world. Stuart Cameron has elegantly stated the position in India and outlines paid donor transplantation - "rewarded gift" where there is careful selection of donor panel, publicly audited accounts and results, non-involvement of the middle man, care of the donor and transplant done in a centre with good results. The "backstreet" style is something that one has to avoid at all costs; the backstreet is what happens in several "centres" in India and is the antithesis to the “rewarded" gift.
With the shortage or organ donors and the ever increasing number of patients requiring transplantation major interest has revived again in xenografting from foreign species. Whilst primates, are ideal they are small in number. Only an animal such as a pig with its multipapillate kidney of about the same size, concentrating ability and proportion of nephrons as a human kidney, is feasible as a suitable source of organ in the future. Before xenografts can be used, there is a need to abrogate the xenograft response. This involves principally antibodies usually, but not always, of IgM isotype, present at birth and not induced by oral immunisation from the carnivorous diet. These so called “natural" antibodies react with antigens expressed on foreign endotherlial cells to cause rapid thrombosis and destruction of any xenograft. A good leal of evidence suggest that if this reaction can be avoided then the reaction arising from recognising xeno MHC may be less strong, or certainly no worse than with allo-antigens. There is a good deal of recent work upon ways of modifying the recipient by removing the “natural" antibody and preventing its resynthesis by immunoadsorption or plasma exchanges. The antigens against which the "natural" antibodies react have not been fully identified but the sequence will surely be available soon. A transgenic pig from whose genome the gene coding for the endothelial targets for the "natural" antibody has been deleted, inactivated, or replaced would be required. The advantage of this type of procedure is that transgenic animals can then be bred in fairly large numbers, which will inherit their suitability as donors for humans.
As an intermediate goal induction of tolerance to human allograft remains. This could be achieved in several ways and certainly there is no magic single mechanism, which stands out as the only approach. Possible approaches include the use of anti CD4 antibodies in order to eliminate a vital second signal needed for immune recognition and without which tolerisation can occur. Transplantation under the cover of anti-CD4 antibody looks a realistic possibility for inducing tolerance. Another way is to return the immune system to an earlier state of development by using total lymphoid irradication for selective area before transplantation. Donor marrow is preserved and then infused at the point when alloreactivity appears. Tolerance has already been induced in humans with this technology.
Finally, a new approach being tested clinically is to use antibodies, which bind to passenger leukocytes in the donor organ prior to transplantation. Use of antibody to a common leukocytes antigen CD45, which is a transmembrance antigen on most cells of the haemopoietic system, has resulted in lessening of rejections. Pre-treatment of human renal allografts with anti-leukocytes common antigen mouse monocionol antibodies (IgG2 a non-lytic complement fixing antibody) showed lower incidence of rejection and improved kidney function related to a control group.
Transplantation is already the optimum treatment for terminal renal failure. Donor organ shortage means that there are a large number of patients on dialysis awaiting this treatment. This has in some countries led to unacceptable unscrupulous practices of live non-related graft donation.
The outcome of graft and patient after transplantation has improved significantly based on a better understanding of immunopathology, immunsuppression and tissue typing. The future is promising and xenografting may well solve the organ shortage but undoubtedly will raise other issues.
[Table - 1], [Table - 2]